A detailed map of restriction endonuclease recognition sites on mouse and human mitochondrial DNA (mtDNA) is being constructed. Hind II, Hpa I and II and Bam I sites are currently under investigation. Methods have been devised for in vitro labelling of mtDNA, production of partial digestion products in high yield, separating the products by agarose gel electrophoresis, and efficient recovery of the DNA from gel slices. The physical map produced will be used to locate significant regions, and will be compared with the restriction enzyme digest products using mtDNA from neoplastic and normal cells. Several new techniques are being developed. One is a method for visualizing restriction endonuclease binding sites in the electron microscope. The other is agarose gel electrophoresis, which is a powerful analytical tool for distinguishing differences in both DNA size and conformation. We are applying the latter method to studying DNA replication, and to studying restriction endonuclease reaction mechanisms. Bibliographic references: H.P. Vosberg, L.I. Grossman and J. Vinograd (1975). Eur. J. Biochem. 55, 79. Isolation and Partial Characterization of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells.